This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We are using cryofixed, freeze-substituted cells to study the morphology of kinetochores and their associated microtubule (MT) in mitotic cells. We have developed successful methods for preparing well-frozen mitotic cells (Morphew and McIntosh, J. Micros 212:21-25). 3-D reconstructions of kinetochores from several species have been obtained using dual-axis tomography. The structures of kinetochore-associated MT ends have been oriented and extracted using the "slicer" tool in IMOD. The walls of most kinetochore MTs flare outward at their plus ends, a morphology characteristic of disassembling MTs in vitro (Mandelkow et al., JCB 114:977, 1991). The degree of flaring has been quantified and found to be similarly distributed at all mitotic stages. These findings suggest that flaring in vivo is not simply indicative of disassembly, but may reflect a different dynamic state that can be controlled by other cellular processes. The regions around kinetochore MTs contain slender fibrils that appear to connect the MT walls in the centromeric heterochromatin. These findings suggest a novel mechanism for the binding of chromosomes to the mitotic spindle.